Optical tweezer systems use a laser beam brought into tight focus to change the gradient forces surrounding dielectric particles, where the radiation pressure traps particles. In early experiments, optical gradient forces were created from a single beam of light used to control and manipulate micrometer-sized particles. For example, a single-mode, TEM00 laser beam was brought to a tight focus at or near the sample's focal plane. By providing a focal region of light into a cell, a laser-based light source was able to provide enough radiation pressure to trap a particle immersed in a fluid medium entering the focal region.
For optical trapping, the diameter of the laser beam should closely or exactly fill, or somewhat overfill, the back pupil of an objective lens. By filling the back aperture of the objective lens, the light converges to a tight, diffraction-limited spot. The photons from the laser spot absorb, scatter, or refract a dielectric sphere with an index of refraction higher than the surrounding medium. The photons' momentum changes, and by Newton's Second law, the rate of change of the deflected rays' momentum results in an equal and opposite rate of change in the particle's momentum. Thus, the force impacted from photons is proportional to the spatial gradient of the light intensity, and a trapped particle acts in the direction of that light.
It has been suggested that optical trapping, or tweezer systems could be applied to biological microparticles, for example see U.S. Pat. No. 6,416,190. Cell separation and phenotypic analysis are a rapidly growing area of biomedical and clinical development. Improved methods of separating a desired cell subset from a complex population permit the study and use of cells that have relatively uniform and defined characteristics. Cell separation is widely used in research, e.g. to determine the effect of a drug or treatment on a targeted cell population; investigation of biological pathways; isolation of transformed or otherwise modified cell populations; etc. One widely used method for cell analysis and separation is flow cytometry, where the cells can be detected by fluorescence or light scattering. However, there are significant disadvantages to the use of flow cytometry. Although a high degree of purity can be achieved, cells are processed in series, i.e. single file through the sorter. Even with high flow rates, it is time-consuming to isolate a sufficient number of cells for clinical applications, since several sorting cycles are required.
Conventional optical tweezer systems use a single laser beam to create a single trap, manipulating a particle at a time. But in order to trap and manipulate multiple particles, multiple beams of light must be used. Current optical tweezer techniques and methodology are not readily extended to create multiple beams, thereby limiting the throughput and potential use in many applications. The present invention provides a novel approach and methodology to create high-density arrays of optical traps.
Relevant Literature
U.S. Pat. No. 6,210,910 describes an optical fiber biosensor array comprising cell populations confined to microcavities. U.S. Pat. No. 6,200,737 is directed to photodeposition methods for fabricating a three-dimensional patterned polymer microstructure on solid substrates using unitary fiber optic arrays for light delivery. U.S. Pat. No. 6,023,540 provides a fiber optic sensor with encoded microspheres. U.S. Pat. No. 5,320,814 describes fiber optic array sensors, apparatus, and methods for concurrently visualizing and chemically detecting multiple analytes of interest in a fluid sample. Fluorescence intramolecular energy transfer conjugate compositions and detection methods are disclosed in U.S. Pat. No. 5,254,477.
Optical trapping is described, e.g. in U.S. Pat. No. 4,893,886, as a single-beam gradient force trap. This force trap consists of a strongly focused light beam which has a near Gaussian transverse intensity profile. The stabilizing effect on the trapped particle arises due to the combination of the radiation pressure scattering and gradient force components, which combine to give a locus of stable equilibrium near the focus of the laser beam. Thus, stabilizing the trapped particle occurs by strongly focusing the light. The majority of currently produced optical tweezer systems create a single or a few tweezers, moving a singular or a few particles at a time. Dual beams of light have been used as optical tweezers to manipulate microscopic objects and cells. Both single and dual-beam traps were used to levitate a microsphere from the bottom of a sample chamber (Ashkin (1991) ASGSB Bull. 4(2):133–46).
Taguchi et. al. (2000) Jpn. J. Appl. Phys. 39:L1302–L1304; and Taguchi et al. (2000) IEICE Trans. Electron. E83-C used single mode optical fibers to trap and manipulate microspheres and cells. Cells were also trapped using a single laser beam from an optical fiber inserted at an angle in a sample chamber. Manipulation of the cell was achieved by using a dual optical fiber arrangement. Lyons and Sonek (1995) Appl. Phys. Lett. 66:1584–6 used a dual single mode fiber optical trap with tapered ends coupled to laser diodes to trap dielectric particles. Axial and transverse trapping was exhibited. Sasaki et al. (1991) Jpn. J. Appl. Phys., 30:L907–L909; and Sasaki et al. (1991) Opt. Lett. 16:1463–5 reported on a repetitive laser scanning method to manipulate and pattern multiple microparticles in solution. The particles were aligned by continuously scanning at 13 to 50 Hz by computer controlled galvano mirrors. Mio et al. (2000) Rev. Sci. Instrum 71:2196–2200 have reported a laser-scanning method to manipulate colloids and biological cells in solution. A single beam scanned at rates as high as 1200 Hz to trap multiple colloids simultaneously. In all the methods mentioned above, one trap was used.
Methods have been proposed for creating an array of traps. Dufresne et al. (1998); Rev. Sci. Instrum. 69:1974–1977; Dufresne et al. (2001) Rev. Sci. Instrum. 72:1810–1816; U.S. Pat. No. 6,055,106; and U.S. Pat. No. 6,416,190 disclose techniques for creating multiple optical tweezers using commercially available diffraction gratings as well as computer-generated holograms. The diffractive optical elements generate triangular and square tweezer arrays with up to 400 individual traps.
Ogura et al. (2001) Appl. Opt. 40:5430–35 propose a method for a trap array using multiple beams generated by a vertical-cavity surface emitting (VCSEL) array. Multiple particles were simultaneously captured and manipulated by using an 8×8 VCSEL-based tweezer array. Mogensen and Gluckstad (2000) Opt. Commun. 175:75–81 report a method of creating an optical tweezer array, by using a phase-only liquid crystal spatial light modulator (SLM) to encode an image directly in the phase component of a laser beam. This general phase contrast approach creates a low loss system to simultaneously manipulate multiple microparticles.